Application of berberine in medicament for treating hepatitis B virus infection
A technology of hepatitis B virus and berberine, applied in the field of medicine, can solve problems such as the severe situation of hepatitis B prevention and treatment, and achieve the effects of improving treatment quality and medication compliance, abundant drug resources, and convenient use.
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Embodiment 1
[0036] Example 1: Cytotoxicity of berberine to HepG2 cells and HepG2 2.15 cells
[0037] Disperse well-grown HepG2 cells and HepG2 2.15 cells into a single cell suspension with trypsin, press 1×10 5 / ml concentration in 96-well plate, 0.1ml per well, set at 37°C, 5% CO 2 Cultivate in the incubator for 24h. After the cells grow into a single layer, the supernatant of the culture medium is discarded and replaced with a drug-containing maintenance solution of different concentrations. Three replicate wells are set for each concentration, and a normal cell control is set. Continue culturing for 48 h, discard the culture supernatant, add 100 μl of serum-free DMEM medium containing 100 ug / ml of MTT (tetramethylazozolium salt) to each well, and place in CO 2 After continuing to culture in the incubator for 2-3 hours, discard the MTT supernatant, wash 3 times with PBS, add 100 μl of dissolving solution (DMSO:ethanol volume ratio: 1:1) to each well, shake for 5-10 minutes, and set th...
Embodiment 2
[0039] The results showed that the drug had no obvious cytotoxicity in the concentration range of 0.32ug / ml-1000ug / ml. Example 2: Effect of berberine on HBsAg and HBeAg in HepG2 2.15 cells in vitro
[0040] 1) Take the coated microwell reaction strip, fix it on the plastic frame, add 50 μl / well of the sample, 50 μl (or 1 drop) / well of the negative control and 50 μl (or 1 drop) of the sex control in each two wells, and leave one well without adding it as a blank control, and then add Measure HBsAg or HBeAg enzyme conjugate 50μl (or one drop) / well, blank control is not added.
[0041] 2) Place a constant temperature water bath at 37°C for 30 minutes.
[0042] 3) Carefully remove the liquid in the wells and pat dry; dilute the washing solution 1:20 times with distilled water, fill each well, and discard after staying for 30 seconds. Repeat this way for 6 times and pat dry each time. Avoid cross-contamination between wells as much as possible.
[0043] 4) Add 50 μl (or one drop...
Embodiment 3
[0061] Example 3: Effects of Drugs on Some Classical Signaling Pathways
[0062] 1. Lipofectamine transfection method:
[0063] The negatively charged DNA binds to the positively charged end of the cationic compound, and then the DNA-binding lipid binds to the cell membrane, allowing the DNA to enter the cell. The liposome transfection method can produce extremely high transfection efficiency for some cells. The liver cells HepG2 used in this experiment (purchased from the China Typical Microorganism Collection Center) have been confirmed by long-term transfection experiments in the laboratory. Dyeing is better. The specific method takes the transfection operation as an example with a 24-well plate. The whole process of transfection must be strictly aseptic.
[0064] 1) Seed the cells to be transfected in a 24-well plate one day before transfection and culture at 37°C. The cell density before transfection was 40-60%.
[0065] 2) Prepare the following solution
[0066] a)...
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